Gel filtration (size exclusion chromatography) and high-performance liquid chromatography (HPLC) have been widely used in separation and purification of soluble proteins. Both methods employ filtration columns, but the column packing materials are different. Most common packing materials are polymers for gel filtration and porous silicon particles for HPLC. Polymers are often preferred for biological applications for the convenience of not requiring any additional chemical modification. It is well-known that in filtrations processes, microscopic pores of the packing material effectively elongate flow paths for soluble proteins and that the net path increase for a protein depends on its molecular weight. Thus, differing proteins elute at differing times in a filtration run, allowing identification and purification according to the elution pattern. With HPLC, usually silicon particles are chemically modified to better facilitate biological applications because the silicon surface is hydrophobic and thus is prone to air bubbling that markedly affects the continuous uniform flow of the transport liquid in filtration runs.
Dynamic light scattering (DLS) is well-known in the context of measuring hydrodynamic sizes, polydispersities and aggregation effects of protein samples. In a conventional DLS setup, a fixed volume of a transport liquid containing the protein samples under investigation is placed in a transparent cell and laser light is shone into the cell. A photodetector is then used to measure the laser light that is scattered from the protein particles suspended in the transport liquid. Fluctuations of the scattering intensity due to the Brownian motion of the particles are recorded and used to determine the sizes, polydispersities and aggregation effects of protein samples according to well-known techniques.